Abstract

In lung cancers, TTF-1 displays seemingly paradoxical activities. Although TTF-1 is amplified in primary human lung cancers, it inhibits primary lung tumors from metastasizing in a mouse model system. It was reported that the oncogenic proepithelial mesenchymal transition (EMT) high mobility group AT-hook 2 gene (HMGA2) mediates the antimetastatic function of TTF-1. To gain mechanistic insight into the metastasis-critical signaling axis of TTF-1 to HMGA2, we used both reverse and forward strategies and discovered that microRNA-33a (miR-33a) is under direct positive regulation of TTF-1. By chromatin immunoprecipitation, we determined that TTF-1 binds to the promoter of SREBF2, the host gene of miR-33a. The 3'-untranslated region (UTR) of HMGA2 contains three predicted binding sites of miR-33a. We showed that the first two highly conserved sites are conducive to HMGA2 repression by miR-33a, establishing HMGA2 as a genuine target of miR-33a. Functional studies revealed that enforced expression of miR-33a inhibits the motility of lung cancer cells, and this inhibition can be rescued by overexpression of the form of HMGA2 without the 3'-UTR, suggesting that TTF-1 keeps the prometastasis gene HMGA2 in check via up-regulating miR-33a. This study reports the first miRNAs directly regulated by TTF-1 and clarifies how TTF-1 controls HMGA2 expression. Moreover, the documented importance of SREBF2 and miR-33a in regulating cholesterol metabolism suggests that TTF-1 may be a modulator of cholesterol homeostasis in the lung. Future studies will be dedicated to understanding how miRNAs influence the oncogenic activity of TTF-1 and the role of TTF-1 in cholesterol metabolism.

Screening murine lung cancer cells for Ttf-1-regulated miRNAs that target HMGA2 using an LNA-QPCR array.A, total RNA isolated from 394T4-shLuc (Ttf-1high) and 394T4-shTtf-1 (Ttf-1low) cells was quantified in duplicate using an LNA-QPCR array designed to include miRNAs predicted to target the 3′-UTR of human HMGA2 gene. Normalized expression values between the two cell lines are expressed as -fold change of shLuc over shTtf-1 cells. B, down-regulation of mmu-miR-33 in 394T4-shTtf-1 cells was confirmed by RT-QPCR using an miRNA detection system and PCR primers (Quanta Biosciences) (n = 3). Error bars, S.D. *, p < 0.05.

A global screen for TTF-1-regulated miRNAs using a microarray platform.A, immortalized human lung epithelial cells with Dox-inducible TTF-1 elements (BEAS-2B-rtTA3-TTF1) were treated with or without dox (0.8 μg/ml) for 24 h prior to protein harvest. Western blot analysis confirmed the induction of TTF-1, with HSP90 protein as the loading control. B, RNA isolated from BEAS-2B-rtTA3-EV or TTF-1 cells treated with or without dox (0.8 μg/ml) for 24 h was evaluated in duplicates for changes in miRNA expression using an LNA-miRNA microarray (Exiqon). Expression of miRNAs between the different groups was compared by log median scores (LMS), and the average miRNA expression changes are graphed as indicated. Lines on the graph indicate the 1st and 99th percentile of the data. C, up-regulation of miR-32 and miR-33a in BEAS-2B-rtTA3-TTF-1 cells was confirmed using RT-QPCR (n = 3). Error bars, S.D. *, p < 0.05; **, p < 0.01.

HMGA2 is a novel target of miR-33a in mouse and human lung cancer cells.A, diagram depicting the 3′-UTR of the human HMGA2 gene. Locations of predicted miRNA binding sites for let-7 (black), miR-33a (gray), and miR-32 (white) are marked with arrows, and seed site locations are listed below the arrows. B, NCI-H1299 cells were transfected with the HMGA2 3′-UTR reporter and either a scrambled control oligonucleotide (Scr) or an miR-33a mimic (miR-33a). Luciferase activity was read 48 h post-transfection (n = 3). C, HMGA2 3′-UTR is not a target for miR-32. NCI-H1299 cells were cotransfected with an miR-32 mimic or a negative control oligonucleotide (NC). At the same time, the transfections include a wild-type HMGA2 3′-UTR reporter construct (Wt) or an HMGA2 3′-UTR reporter with a deleted miR-32 binding site (ΔmiR-32). After 48 h, Renilla and firefly luciferase activities were assayed. Although miR-32 resulted in a slight inhibition of the HMGA2 reporter, deletion of the sole predicted miR-32 binding site did not cause a derepression of the HMGA2 reporter. RLU, relative luminescence units. D, quantification of endogenous HMGA2 mRNA after transfection of NCI-H1299 cells with a scrambled control oligonucleotide or an miR-33a mimic. HGMA2 expression levels were normalized to GAPDH (n = 3). E, Western blot analysis confirmed a reduced expression of HMGA2 in the human NCI-H1299 cells transfected with miR-33a mimic. F, the Hmga2 expression was knocked down in the murine 394T4-shTtf-1 cells transfected with an miR-33a mimic. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

TTF-1 up-regulation of miR-33a suppresses HMGA2 expression in human and mouse lung epithelial cells.A, Western blot analysis of Hmga2, Ttf-1, and Hsp90 expression in murine 394T4-shLuc cells transfected with either a negative control (anti-miR-NC) or an miR-33a inhibitor (anti-miR-33a). B, Western blot analysis of HMGA2, TTF-1, and Hsp90 expression in the human NCI-H358 cells transfected with either a negative control or an miR-33a inhibitor. C, 394T4-shTtf-1 cells were transfected as indicated, and the corresponding cell lysates were analyzed by immunoblotting for the expression of Ttf-1, Hmga2, and Hsp90.

Knockdown of endogenous Ttf-1 and exogenous miR-33a slow migration but do not alter invasiveness.A, murine 394T4-shLuc and shTtf-1 cells were allowed to migrate through a membrane with 8-μm pores (Migration) or a membrane with 8-μm pores coated with Matrigel (Invasion) for 22 h. B, data from A were used to calculate the percentage invasion (% invasion = (mean number of cells invaded through the Matrigel-coated membrane)/(mean number of cells invaded through the uncoated membrane)). C and D, experiments here were conducted as in A and B except that NCI-H1299 cells transfected with a negative control oligonucleotide (NC) or an miR-33a mimic oligonucleotide were analyzed. Error bars, S.D. *, p < 0.05; ***, p < 0.001.